The goal of this project is to develop strategies for achieving clinically relevant levels of gene transfer into hematopoietic stem cells for the purpose of treating inherited disorders of hematopoietic stem cells and their progeny. To date, in vitro stimulation alone, even with multiple growth factors, has not resulted in enhanced gene transfer into hematopoietic stem cells. We hypothesize that, as in mice, it will be necessary to induce stem cell proliferation to achieve therapeutic levels of gene transfer. In mice it is essential to treat animals with 5-FU prior to harvesting marrow to achieve efficient gene transfer into marrow repopulating stem cells. We hypothesize that treatment with cytokine combinations and/or cytotoxic drugs to perturb marrow will elicit proliferation of stem cells, thereby rendering them more susceptible to retroviral or AAV mediated gene transfer. In the course of these studies we will compare CD34+ stem cells from different sources for susceptibility to transduction by different vectors. We will determine if treating animals with hematopoietic growth factors prior to harvesting marrow and blood CD34+ stem cells will alter gene transfer into marrow repopulating cells. We will determine the ability of genetically altered CD34+ cells from different sources (marrow, blood, cord blood) to reconstitute lymphohematopoiesis in vivo. These studies to define the conditions necessary to achieve effective gene transfer into baboon hematopoietic stem cells will provide further insights that will be directly applicable to gene therapy in humans.